Single-molecule fluorescence resonance energy transfer (smFRET) has rapidly developed to answer fundamental questions about biological mechanisms, providing detailed views of molecular machines at work. Nesher Technologies, Inc. (NTI) was established to commercialize an innovative quantitative, ultrasensitive and -specific single-molecule biodetection technology with exquisite multiplexing potential and simplified sample handling procedures. It was pioneered at the UCLA Single Molecule Biophysics Lab, which is headed by Prof. Shimon Weiss. The technology is based on alternating-laser excitation (ALEX) of single molecules labeled with fluorescent probes. In addition to its suitability for diagnostics, ALEX has been employed recently to study the dynamics of molecular processes such as transcription initiation and elongation using smFRET. In two-color (2c)-ALEX, employing two alternating lasers to study molecular interactions (through probe stoichiometry ratio S and/or FRET efficiency E) and intramolecular distances for analysis of conformation and mechanism (through E), molecules are sorted in a two-dimensional histogram of S and E. In 3c-ALEX, molecules are sorted in three-dimensional S and E histograms, substantially extending the capabilities of 2c- ALEX. ALEX can be performed with diffusing molecules, enabling analysis of equilibrium behaviors of single molecules, or with immobilized molecules using millisecond-scale ALEX dynamic imaging (ALEX-DI). The ALEX-DI methodology, employing total-internal-reflection optical microscopy, has the advantage over its sister methodology, confocal microsecond-scale ALEX spectroscopy, that non-equilibrium time trajectories can be established to follow kinetics of individual molecules and complexes. Detailed stochastic molecule information on complex composition and function can be obtained as well. The ability to monitor individual molecules for long stretches of time adds a whole new dimension with dynamic information ranging from milliseconds to minutes. Higher-order FRET schemes can also be applied to probe transient multi-component interactions or spatiotemporal relationships between different conformational changes in large molecular complexes. NTI's long-term goal is development of a fully integrated 5-color ALEX-based instrument suitable for transient molecular complex characterization. The high-resolution power to monitor molecular interactions and conformational dynamics will also greatly aid structure-guided rational drug design endeavors. For Phase I, we propose to extend 2-color ALEX-DI to enable 3-color and 4-color functionality. Specific aims are: 1. Construction of an instrument for single-molecule total-internal-reflection optical microscopy with 4-color alternating-laser-excitation and dynamic imaging (4c-ALEX-DI). 2. Preparation of fluorophore-labeled bacterial transcription initiation complex components. 3. Use of 4c-ALEX-DI in 3-color and 4-color mode to detect and characterize DNA-scrunching in immobilized RNA polymerase (RNAP)-promoter initial transcribing complexes (RPitc), as well as promoter escape. PUBLIC HEALTH RELEVANCE: The proposed multi-color alternating-laser excitation (ALEX)-based single-molecule method for identifying and characterizing transient molecular complexes will vastly improve the ability to gather molecular information, which in turn will enhance our ability to understand the complex effects that subtle mutations and/or environmental factors have in the development and progression of diseases. The ALEX technology, highly useful for basic research as well as drug development applications, can complement structural analysis of biomolecules and their complexes, especially for species intractable by conventional structural biology methods due to excessive heterogeneity, limited stability, large size, presence of flexible domains, and/or transient nature. As an example, the proposed instrumentation will be able to facilitate detailed mechanistic studies of the bacterial transcription process, allowing rapid development of next-generation antibiotics (e.g. inhibitors of bacterial transcription at new sites of the transcription machinery), which is of critical importance for effective control of globally emerging multi-drug resistant bacterial strains, such as multidrug-resistant tuberculosis (MDR TB), extensively drug-resistant tuberculosis (XDR TB), methicillin-resistant Staphylococcus aureus (MRSA),and vancomycin-resistant enterococci (VRE).